Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/38—Diluting, dispersing or mixing samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1434—Optical arrangements
  • G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
  • G01N35/1097—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers characterised by the valves
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
  • G01N2015/011—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells with lysing, e.g. of erythrocytes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N2035/1027—General features of the devices
  • G01N2035/1032—Dilution or aliquotting

Definitions

• the present invention relates to a method for dilution of a sample for analysis and to a haematology apparatus for implementation of such a method.
• This sample may be blood or other biological liquid such as, for example a puncture fluid such as a cerebrospinal fluid (CSF) containing white blood cells or red blood cells.
• CSF cerebrospinal fluid
• a haematology apparatus makes it possible to count and characterize different types of cells present in the blood.
• the document US 6,333,197 (ABX) is also known, describing a needle for collecting blood and injecting it into different chambers at the same time as a reagent to produce a homogenous dilution.
• the system described in this document US 6,333,197 requires collection of a large quantity of whole blood which by design is not totally used.
• the positioning of the needle in the different chambers is complex due to the requirement for the alignment of the needle with the arrival of the reagent.
• the chambers are specifically designed in order to allow the homogenization of the blood with the reagent.
• An object of the present invention is a novel dispensing method that is rapid and simple to implement.
• Another object of the invention is a novel method using a small quantity of whole blood for characterizing the blood cells, for example the white blood cells and the reticulocytes. At least one of the above-mentioned objectives is achieved with a method for dilution of a blood sample for analysis, this method comprising the following steps:
• steps a) to e) at least one analysis of the first-dilution liquid and/or second-dilution liquid and/or third-dilution liquid.
• This single collection can, for example, be a quantity of 20 pi of whole or diluted blood, whereas in the prior art this collection is generally of the order of 120 mI or more.
• This method has the advantage of being simple to implement because the retention of the first- dilution liquid in the aliquoting device for the second and the third dilutions is cleverly used.
• the method according to the invention allows high analysis speeds.
• the second- and third-dilution liquids are obtained independently of one another, i.e. the third-dilution liquid is not obtained from the second-dilution liquid but directly from the first-dilution liquid retained in the aliquoting device.
• the dilutions can take place successively in a single chamber or in several chambers.
• the analyses can take place successively or simultaneously (in parallel) depending on the chosen configuration with one chamber or several chambers.
• the analysis can comprise characterizing the first and/or second and/or third dilution liquid by optical measurement for counting and/or differentiating particles contained in the liquid.
• the analysis can comprise counting particles in the first- and/or second- and/or third-dilution liquids, by means of a resistive sensor.
• the optical measurement can take place on an optical bench or in the chamber used for the dilution, this chamber then being equipped with an optical device.
• one or more resistive sensors can be connected to or incorporated in at least one chamber or in an optical bench.
• optical bench is meant a device making it possible to:
• step e) can comprise the following steps:
• el injecting into a chamber, preferably into a second chamber a first quantity of the first-dilution liquid contained in the aliquoting device, a second quantity of the first-dilution liquid remaining in the aliquoting device, e2) diluting the first-dilution liquid contained in the second chamber, by means of a dilution reagent so as to constitute a second-dilution liquid, e3) injecting a lysis solution into the first chamber to destroy red blood cells,
• At least one chamber preferably two chambers, and one optical bench, it is possible to carry out a set of counting and/or differentiation measurements.
• the analysis speed is very high.
• step elO it is possible to transfer to the optical bench a portion of the third- dilution liquid for differentiation of the red blood cells, in particular the immature red blood cells, the reticulocytes.
• a single sample collection allows differentiation of the white blood cells and differentiation of the red blood cells.
• an aliquoting device comprising:
• a needle capable of being moved between a sample collection zone and said at least one chamber, - a dilution reagent dispenser, and
• a sampling valve comprising at least two liquid pathways and a calibrated-volume channel, a first liquid pathway linking the dispenser to the needle, a second liquid pathway linking the dispenser to the second chamber, and the calibrated-volume channel activating the first liquid pathway or the second liquid pathway.
• a sampling valve can be designed comprising two ceramic discs, one of which contains the calibrated-volume channel. This channel can be shifted between two positions, a first position where the channel is comprised within the first liquid pathway and a second position where the channel is comprised within the second liquid pathway.
• the invention is in particular remarkable for the re-use of the first dilution present in the needle up to the sampling valve.
• Step d) can preferably be carried out by collecting the sample and retaining it inside the needle and in the calibrated-volume channel of the sampling valve.
• Step d) can preferably be carried out by collecting the sample and retaining it inside the needle and in the calibrated-volume channel of the sampling valve.
• the sampling valve forms part of the fluidics circuit for collecting the first dilution.
• the needle makes it possible to collect the first dilution in the first chamber, but injection into the second chamber is carried out directly via the second liquid pathway. More precisely, a tube makes it possible to link the sampling valve to the second chamber. This feature makes it possible, for example, to carry out the second dilution while retaining a portion of the first dilution in the needle, which makes it possible to subsequently dispense a portion of it into the first chamber for the third dilution without being obliged to carry out a new collection of the sample.
• step d) can be carried out by collecting the first-dilution liquid and retaining it inside the needle and in the sampling valve.
• the first-dilution liquid is preferably aspirated into the sampling valve and beyond this valve into a tube between the sampling valve and the dispenser.
• an aliquoting device comprising one or more sets of precision pistons/syringes in order to collect and inject the sample and the different dilutions from and into the different chambers.
• the volumes injected in order to carry out the first dilution and the third dilution are determined by precisely controlling the piston(s)/syringe(s).
• the steps of the first and second dilutions can preferably be carried out by injecting a reagent of dilution via the aliquoting device.
• the aliquoting device comprises a needle and the sampling valve
• the two liquid pathways comprise tubes in which the dilution reagent originating from the dilution reagent dispenser serves as liquid for dispensing the sample and/or dilution reagent.
• all or some of the dilution steps can be carried out by injecting dilution reagent from a liquid pathway independent of the aliquoting device and directly into the chamber or chambers.
• a single optical bench linked to the first chamber can be used.
• all or some of the counts are carried out by means of resistive sensors connected to the first and/or second chamber and/or to other chambers if there are more than two chambers.
• the first dilution can have a ratio of 1/200
• the second dilution can have a ratio of 1/10,000
• the third dilution can have a ratio of 1/10,000.
• the injection of the lysis solution in step e3) can be carried out via a liquid pathway independent of the aliquoting device and directly into the first chamber.
• This lysis solution has the function of destroying the red blood cells and separating the white blood cells. This also allows stabilization of the haemoglobin in the form of a stable complex.
• the method can comprise a step of adding a fluorescent dye to the first chamber before each optical differentiation measurement.
• An optical bench making it possible to detect the fluorescence can preferably be used. It is thus possible to detect the reticulocytes, immature red blood cells, thanks to the presence of the fluorescent dye.
• a fluorescent dye to the first- and/or second- and/or third-dilution liquid before any optical measurement, so as to improve the differentiation of the blood cells for example the white blood cells or/and the characterization of the reticulocytes using fluorescence.
• steps e5) and e6) can be carried out in parallel or sequentially.
• the counts in parallel are carried out using a single aspiration system, allowing aspiration from both chambers into different channels at the same time. It is perfectly possible to envisage separate (non-simultaneous) counts with a single or several distinct aspiration systems.
• a haematology apparatus for the automatic counting and differentiation of cells in a blood sample characterized in that it comprises:
• a needle capable of being moved between a sample collection zone and at least one chamber
• a sampling valve comprising at least two liquid pathways and a calibrated-volume channel, a first liquid pathway linking the dispenser to the needle, a second liquid pathway linking the dispenser to at least one chamber, and the calibrated-volume channel activating the first liquid pathway or the second liquid pathway.
• a treatment unit for implementing the different steps and controlling the different components is also provided.
• the sampling valve according to the invention can contain a calibrated- volume channel, this calibrated-volume channel being capable of constituting either a part of the first liquid pathway or a part of the second.
• the calibrated-volume channel switches over from one liquid pathway to the other.
• the dispenser can control the aspiration or the expulsion of a portion of the liquid contained in the first liquid pathway, the second liquid pathway being non-operational.
• the dispenser can control the expulsion of a portion of the liquid contained in the second liquid pathway, the first liquid pathway then being non-operational.
• Figure 1 is a diagrammatic view illustrating a few components constituting an automatic haematology analyser that is ready for use
• Figure 2 is a diagrammatic view illustrating a preliminary step of whole blood collection
• Figure 3 is a diagrammatic view illustrating a step 1 of constituting a first dilution
• Figure 4 is a diagrammatic view illustrating a step 2 of collecting a portion of the first dilution
• Figure 5 is a diagrammatic view illustrating a step 3 of constituting a second dilution
• Figure 6 is a diagrammatic view illustrating a step 4 of transfer to an optical bench for a differentiation of white blood cells
• Figure 7 is a diagrammatic view illustrating a step 5 of emptying and rinsing the chambers
• Figure 8 is a diagrammatic view illustrating a step 7 of constituting a third dilution
• Figure 9 is a diagrammatic view illustrating a step 8 of transfer to the optical bench for a differentiation of red blood cells
• Figure 10 is a diagrammatic view illustrating a step 9 of emptying and final rinsing.
• Figure 1 illustrates components constituting an automatic haematology analyser that is ready for use, awaiting an analysis cycle.
• An optical bench 1 for characterizing different types of cells present in the blood can be seen.
• a first chamber 2 is linked to the optical bench 1 via a solenoid valve 3 capable of blocking or allowing the passage of fluid contained in the first chamber 2 to the optical bench 1.
• the first chamber 2 comprises an outlet 21 connecting to the solenoid 3, and electronic means, in particular at least one sensor 22, for resistivity measurements. These measurements are, for example, implemented during cell counts.
• optical bench 1 For the sake of clarity of the diagram, only the optical bench 1 is shown; it is clear that a flow cell (not shown) is provided within this optical bench, in which the fluid to be characterized can flow.
• a dilution reagent dispenser 4 can also be seen, linked to a sampling valve 5 via two parallel conduits Cl and C2.
• the sampling valve 5 is linked on one side to a needle 6 via a conduit C3 and on the other side to a second chamber 7 via a conduit C4.
• the sampling valve 5 is a valve comprising two liquid pathways and a calibrated-volume channel 8.
• the first liquid pathway makes it possible to link the conduits Cl and C3 via the calibrated-volume channel 8.
• the second liquid pathway makes it possible to link the conduits C2 and C4 via the calibrated-volume channel 8.
• This calibrated-volume channel can thus form part of the first liquid pathway or of the second liquid pathway but not both at the same time.
• this calibrated-volume channel 8 is a conduit suitable for switching from one liquid pathway to the other and forms a reservoir of fluid, the volume of which is very precisely predetermined. A predetermined quantity of liquid can thus be sent from one liquid pathway to the other.
• the conduit C4 is connected to the second chamber 7 via an inlet 71.
• This second chamber 7 also comprises electronic means, in particular at least one sensor 72, for resistivity measurements. These measurements are, for example, implemented during cell counts.
• An independent liquid pathway 74 can also be provided for injecting dilution reagent.
• a treatment unit 9 capable of controlling the different components can also be seen.
• blood is collected in the needle 6 from a tube of whole blood 10.
• a certain volume of blood is then situated only in a part of the needle.
• the first liquid pathway comprising the conduits Cl and C3 is mainly filled with dilution reagent, except for the part of the needle 6 containing blood. It is via an aspiration function via the dilution reagent dispenser that the needle collects the blood.
• the first chamber 2 is emptied.
• step 1 in Figure 3 the needle 6 is moved as far as into the first chamber 2 so as to inject all of the collected blood into it. And the injection is continued so as to fill up with the dilution reagent contained in the first liquid pathway and delivered via the dispenser.
• the mixture of the blood thus deposited with a volume of dilution reagent much greater than the volume of collected blood constitutes the first-dilution liquid with a ratio, for example, of one volume of blood to two hundred volumes of dilution reagent.
• step 2 in Figure 4 a portion of the first dilution is collected from the first chamber 2 as far as into a part of the conduit Cl. Consequently, the needle 6, the conduit C3 and the sampling valve, in particular the calibrated- volume channel 8, are completely filled with the first-dilution liquid.
• step 3 in Figure 5 the calibrated-volume channel 8 filled with first- dilution liquid is switched from the first liquid pathway to the second liquid pathway; the latter is now operational.
• the fact of having aspirated in the step 2 the first-dilution liquid as far as into a part of the conduit Cl made it possible to completely fill the calibrated-volume channel 8.
• a second-dilution liquid is thus formed with a ratio, for example, of one volume of clean blood to ten thousand volumes of dilution reagent.
• the needle 6 is raised again so that it does not remain in contact with the liquid in the first chamber 2 and the lysis solution is injected into this first chamber 2 via an inlet 23.
• the lysis solution has the function of destroying the red blood cells.
• the first-dilution liquid remains present in the needle 6 and in a part of the first liquid pathway comprising the conduit Cl and the conduit C3.
• step 4 in Figure 6 the solution is transferred from the first chamber 2 to the optical bench for differentiation of the populations of the white blood cells.
• the white blood cells are counted in the first chamber 2 by measuring resistivity and a haemoglobin measurement is carried out by means of a spectrophotometer (not shown).
• the red blood cells and platelets are counted by measuring resistivity.
• the counting in the second chamber 7 can be carried out simultaneously with the counting in the first chamber. This is particularly the case when a single aspiration system (not shown) is used for both chambers during the counting process.
• the counting sequence requires to aspirate, by means of the generation of a vacuum, the liquid contained in the chamber through a calibrated orifice based on the impedance measurement principle.
• step 5 in Figure 7 the two chambers are rinsed and emptied completely, as is the fluid circuit between the first chamber 2 and the optical bench 1. It is possible to use the dilution reagent for rinsing the chamber, in order to send it in the fluid circuit to the optical bench and thus to rinse and refill this circuit.
• the calibrated-volume channel 8, filled with dilution reagent, is then switched to the first liquid pathway.
• step 7 in Figure 8 a third dilution is carried out according to the invention.
• a quantity of first-dilution liquid still present in the needle 6 is injected into the first chamber 2.
• a specific volume is pushed.
• a residual first-dilution volume can still be present in the needle 6.
• the dilution is carried out by injecting dilution reagent via an inlet 24 of the first chamber 2.
• the supply circuit of this inlet 24 from the dispenser 4 is not shown.
• a fluorescent dye can also be added.
• step 8 in Figure 9 the solution is transferred from the first chamber 2 to the optical bench 1; then the differentiation of the red blood cells and the reticulocytes is carried out.
• step 9 in Figure 10 when the differentiation is completed, the needle 6 is emptied of residual blood.
• the chambers are rinsed then refilled with dilution reagent, awaiting a subsequent analysis.
• the invention thus makes it possible to perform several differentiation measurements based on a single collection, cleverly using an aliquoting device that allows a first-dilution liquid and the dilution reagent to be segmented.
• the present invention thus relates to a method for dilution of a blood sample for analysis and to an apparatus for implementation of such a method.
• an aliquoting device is used, making it possible to carry out a single collection, to form a first dilution in a chamber, to collect a portion of the first dilution in order to form a second dilution in another chamber, to count the blood cells in the first and the second chamber, to carry out a differentiation based on the first dilution, to rinse the first chamber, to form a third dilution starting from a quantity of first-dilution liquid remaining in the aliquoting device, then to carry out a differentiation of reticulocytes based on this third dilution.
• the invention is not limited to the examples which have just been described, and numerous adjustments can be made to these examples without exceeding the scope of the invention.

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• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
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• Health & Medical Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Dispersion Chemistry (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Sampling And Sample Adjustment (AREA)

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• 2019
  • 2019-07-26 US US17/597,505 patent/US12385816B2/en active Active
  • 2019-07-26 CN CN201980098746.1A patent/CN114174796B/zh active Active
  • 2019-07-26 PL PL19780341.4T patent/PL4004521T3/pl unknown
  • 2019-07-26 ES ES19780341T patent/ES3036157T3/es active Active
  • 2019-07-26 CA CA3145364A patent/CA3145364A1/en active Pending
  • 2019-07-26 JP JP2022505393A patent/JP7400073B2/ja active Active
  • 2019-07-26 WO PCT/IB2019/000795 patent/WO2021019267A1/en not_active Ceased
  • 2019-07-26 EP EP19780341.4A patent/EP4004521B1/de active Active

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